The myofibril networks in the mature heart must be highly organized to generate the systemic blood flow effectively. The positions of both the myofibrils and the fasciae adherentes into which the myofibrils terminate must therefore be precisely fixed in space. Thus, how the myofibril-fascia-plasmalemma relationship is established is a fundamental question in understanding the developmental process of cardiac muscle organization, in the chick embryo heart, myofibrils first appear at H-H stage 10 (1.5 days old) and the systemic blood flow is initiated at stage 12+ (2 days old). During the past two years of research supported by this grant, it was found that highly complex myofibril networks traversing through a large number of myocytes are already present in the earliest functional hearts of 2-3 day embryos. One aim of this proposed work is to determine how such complex networks are formed in as short a period of time as 12 hrs. More specifically, the questions to be answered are: 1) how are the fasciae adherentes formed?; ii) do the orientations of the myofibrils determine the positions of the fasciae adherentes or vice versa?; iii) how do functioning myofibrils elongate at the same time as they interact with the fasciae adherentes?; and iv) where does the growth of the myocardial wall most actively take place? In coincidence with the initiation of the systemic blood flow, myocyte networks begin to form trabeculae. Intertrabecular spaces thus created serve as sinusoids for myocardial blood circulation. The sinusoid system which is essential for the contraction and development of the early embryonic heart is later switched to the more efficient capillary system. Thus, another aim is to study this switching process which must occur without ever interrupting myocardial circulation. One possible switching mechanism to be considered is that capillaries grow not into intertrabecular spaces, as previously thought by many investigators, but into intermyocyte spaces in the trabeculae so that the sinusoids continue to function as routes for myocardial circulation during the development of the capillary networks. In order to visualize or identify the structures which are the subjects of these studies, full advantage will be taken of immunocytochemical approaches, in particular, the immunocytochemical labeling methods of semi-thin and ultrathin frozen sections, utilizing antibodies to their component proteins. Double immunolabeling techniques will be used to study the relationship between different pairs of these proteins.